The long-range goal of this research project is to enable the production of innovative sets of molecules for the pretargeted diagnosis and therapy of cancer. The present proposal involves a collaborative effort that seeks to maximize the cure rate of relapsed B cell lymphomas using radiotherapy targeted by engineered antibodies. The focus of this application is pretargeting, which separates the slow accumulation of an antibody in a tumor from the delivery of radiation. We will do this by designing and expressing new fusion proteins prepared from the genes of an antitumor antibody and a ligand-binding protein. After the fusion protein has been administered, localized in the tumor and cleared from the circulation, a small ligand linked to a radionuclide can be captured quickly by the ligand-binding protein pretargeted at the tumor. Using fundamental chemical principles, our lab has developed antibody/ligand pairs that retain the binding specificity of the antibody, but do not dissociate. By eliminating the dissociation of the ligand from the antibody, to produce an irreversible antibody (iAb), we have made the affinity functionally infinite. Irreversible binding in a humanizable format offers striking improvements over the streptavidin-biotin system currently used for pretargeting applications. The work proposed here is a collaborative investigation of irreversible antibodies for the pretargeted delivery of radiotherapy in vivo. Our lab will design, prepare, and characterize new engineered proteins incorporating both antitumor and irreversible anti-ligand binding activity. Our collaborators in Dr. Press' laboratories will use a mouse model system to study the properties of these new iAb constructs for tumor pretargeting, in comparison with conventional radioimmunotherapy using radiolabeled antitumor antibodies and with streptavidin-biotin pretargeting approaches.